contrasting patterns of polymorphism and selection in

Contrasting patterns of polymorphism and selection inbacterial-sensing toll-like receptor 4 in two house mousesubspeciesAlena Fornuskova1,2,3, Josef Bryja1,2, Michal Vinkler4, Milo�s Machol�an5 & Jaroslav Pi�alek1

1Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno, Czech Republic2Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic3Centre de Biologie pour la Gestion des Populations (CBGP), Institut National de la Recherche Agronomique (INRA), Campus International de

Baillarguet, Montferrier-sur-Lez, France4Department of Zoology, Faculty of Science, Charles University in Prague, Prague, Czech Republic5Laboratory of Mammalian Evolutionary Genetics, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Brno,

Czech Republic

Keywords

Adaptive evolution, arms race, directional

selection, host–pathogen interaction,

MAMPs, Mus musculus, parasite-mediated

selection, pattern-recognition receptors.

Correspondence

Alena Forn�uskov�a

Institute of Vertebrate Biology, Academy of

Sciences of the Czech Republic, Research

Facility Studenec, 675 02 Studenec 122,

Czech Republic.

Tel: +420 560 590 621;

E-mail: [email protected]

Funding Information

This research was supported by the Czech

Science Foundation (project 206/08/0640)

and by Ministry of Education, Youth and

Sports of the Czech Republic,

NextGenProject: Next-generation

technologies in evolutionary genetics

(CZ.1.07/2.3./20.0303).

Received: 13 January 2014; Revised: 9 May

2014; Accepted: 14 May 2014

doi: 10.1002/ece3.1137

Abstract

Detailed investigation of variation in genes involved in pathogen recognition is

crucial for understanding co-evolutionary processes between parasites and their

hosts. Triggering immediate innate response to invading microbes, Toll-like

receptors (TLRs) belong presently among the best-studied receptors of verte-

brate immunity. TLRs exhibit remarkable interspecific variation and also intra-

specific polymorphism is well documented. In humans and laboratory mice,

several studies have recently shown that single amino acid substitution may sig-

nificantly alter receptor function. Unfortunately, data concerning polymorphism

in free-living species are still surprisingly scarce. In this study, we analyzed the

polymorphism of Toll-like receptor 4 (Tlr4) over the Palearctic range of house

mouse (Mus musculus). Our results reveal contrasting evolutionary patterns

between the two recently (0.5 million years ago) diverged house mouse subspe-

cies: M. m. domesticus (Mmd) and M. m. musculus (Mmm). Comparison with

cytochrome b indicates strong directional selection in Mmd Tlr4. Throughout

the whole Mmd western Palaearctic region, a single variant of the ligand-bind-

ing region is spread, encoded mainly by one dominant haplotype (71% of

Mmd). In contrast, Tlr4 in Mmm is much more polymorphic with several

haplotypes at intermediate frequencies. Moreover, we also found clear signals of

recombination between two principal haplogroups in Mmm, and we identified

eight sites under positive selection in our dataset. Our results suggest that

observed differences in Tlr4 diversity may be attributed to contrasting parasite-

mediated selection acting in the two subspecies.

Introduction

Selective forces imposed by parasites can affect various

traits of their hosts, including population dynamics,

life histories, mating systems, sexual dimorphism etc.

(Schmid-Hempel 2011). The detrimental effects of para-

sites are countered by function of immune system, which

in vertebrates comprises both innate and acquired immu-

nity (Danilova 2006). Study of evolution in immune-

related genes is, therefore, of paramount importance for

comprehension of dynamics in parasite–host relationships(see e.g., Woolhouse et al. 2002; Carlton 2003). Despite

the complexity of the immune system, most studies in

free-living vertebrates have focused on genes involved in

acquired immunity, namely the major histocompatibility

complex (MHC; e.g., Milinski 2006). However, mapping

and association studies have revealed that at least half of

the genetic variation responsible for resistance to various

ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use,

distribution, and reproduction in any medium, provided the original work is properly cited.

1

infections is attributable to non-MHC genes (Acevedo-

Whitehouse and Cunningham 2006). Most of these genes

seem to be associated with innate immunity and there is

an increasing evidence that variation in these genes may

have a fundamental effect on the host fitness in free-living

populations (e.g., Turner et al. 2011; Tschirren et al.

2013).

Innate immunity receptors that directly detect and bind

to parasite structures (microbe-associated molecular pat-

terns, MAMP), the pattern-recognition receptors (PRR),

stand in the first line of immune defense (Medzhitov and

Janeway 2002; Akira et al. 2006). Their fast and effective

functioning is thus crucial for host survival (O’Neill 2004;

Akira et al. 2006). Among PRRs, the Toll-like receptors

(TLR) have been shown to be particularly important

(Akira et al. 2001). These receptors form a group of

membrane-bound, noncatalytic proteins present in most

immune cells, especially in macrophages. Distinct MAMPs

(e.g., lipopolysaccharides [LPS] and lipoproteins in bacte-

rial cell walls, zymosan of yeast, bacterial flagellin or viral

nucleic acids) are recognized by distinct TLRs and the

set of TLR types varies substantially among vertebrate

lineages (Janssens and Beyaert 2003; Akira et al. 2006;

Vinkler and Albrecht 2009; Kawai and Akira 2010). The

potential action of TLRs in the context of host–parasiteinteractions in free-living organisms is increasingly draw-

ing attention of evolutionary biologists and immunolo-

gists (Medzhitov et al. 1997; Pasare and Medzhitov 2004;

Takeda and Akira 2005; Vinkler and Albrecht 2009).

Contradicting the previous assumption of evolutionary

conservatism of these receptors, evolution-focused immu-

nogenetic investigations yielded a clear evidence that at

the interspecific level diversifying selection has signifi-

cantly increased diversity of orthologous Tlr genes, mainly

in the ligand-binding region (LBR, Poltorak et al. 1998;

Smirnova et al. 2000; Downing et al. 2010; Park et al.

2010; Wlasiuk and Nachman 2010; Areal et al. 2011;

Tschirren et al. 2011; Fornuskova et al. 2013).

Information regarding the structure and variation of

TLRs in free-living rodents is still relatively scarce. Inter-

specific comparisons of European and Asian rodents con-

firmed purifying selection as a prevalent evolutionary

force shaping these genes (namely Tlr2, 4, and 7), proba-

bly due to functional constraints posing on the receptor

molecules (Tschirren et al. 2011; Fornuskova et al. 2013).

However, signatures of positive selection have also been

revealed in all studied genes (mainly in the extracellular

domain [ECD], containing LBRs responsible for pathogen

recognition, see below), with a more intense signal in the

bacterial-sensing Tlr2 and Tlr4 than in the viral-sensing

Tlr7 gene (Tschirren et al. 2011; Fornuskova et al. 2013).

Following study of Tschirren et al. (2012) showed that

TLRs are polymorphic even within species and that

intraspecific variation may strikingly differ even between

two sympatric species of rodents inhabiting the same

environment. In one of these species, the bank vole

(Myodes glareolus), a particular group of alleles was

shown to be significantly associated with resistance to

Borrelia infection, suggesting an on-going evolution in the

receptor (Tschirren et al. 2013). These results illustrate

the urgent need for further research focused on polymor-

phism in PRRs at the intraspecific level, as the genetic

variability in PRRs might represent an important missing

element for understanding the effects of a host genotype

on individual fitness.

TLR4 is one of the most essential bacterial-sensing

PRRs, binding, among others, bacterial endotoxins (i.e.,

LPS) as ligands (Poltorak et al. 1998). At the interspecific

level, this cell-surface receptor has the highest number of

positively selected sites among all mammalian TLRs

(Areal et al. 2011). Most of these sites are localized in the

ECD which is responsible for LPS binding (Poltorak et al.

1998; Kim et al. 2007; Vinkler et al. 2009; Fornuskova

et al. 2013). This domain consists of several leucine-rich

repeat motifs and includes the LBR, which is in direct

physical contact with MAMP structures. The ECD is fol-

lowed by the transmembrane domain (TMD), anchoring

the receptor into the cell membrane, and the intracellular

domain (ICD). The ICD comprises the Toll/interleukin-1

(TIR) domain responsible for signal transduction and cell

activation triggering the immune responses (Werling et al.

2009; Botos et al. 2011).

Genetic research in laboratory mice enabled identifica-

tion of the Tlr4 gene function and assessment of the level

of its polymorphism among laboratory strains (Poltorak

et al. 1998; Smirnova et al. 2000; Stephan et al. 2007).

However, artificial genetic variation occurring in “classi-

cal” laboratory strains (Yang et al. 2011) hampers under-

standing variation present in wild mice displaying much

wider ranges of immunoresponsivity (Pi�alek et al. 2008;

Abolins et al. 2011; Babayan et al. 2011; Pedersen and

Babayan 2011; Riley and Viney 2011). Several house

mouse subspecies have been described. Divergence of

house mice is usually located to northern India and/or

Pakistan and dated to about 0.5 million years ago (Bour-

sot et al. 1993; Suzuki et al. 2004; Geraldes et al. 2008;

Machol�an et al. 2012). Two subspecies, M. m. musculus

(Mmm) and M. m. domesticus (Mmd), have colonized

Europe where they met along a secondary hybrid zone

running across the continent (Boursot et al. 1993;

Machol�an et al. 2007; Bonhomme et al. 2011; Cucchi

et al. 2012, 2013). Although the two subspecies might

come into contact at least once during the expansions

and contractions of their ranges (Duvaux et al. 2011),

allowing them to exchange beneficial mutations, they

remained for most of the colonization time in allopatry.

2 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

Contrasting Patterns of Polymorphism A. Fornuskova et al.

As their westward expansions followed different routes

(Mmm north of the Black Sea, Mmd through the Middle

East and Mediterranean region), the two subspecies may

have experienced different histories leaving distinct

genetic footprints in PRR genes, including Tlr4. A recent

study of the gastrointestinal tract microbiota in western

European mouse populations showed geography to be the

most significant factor explaining the composition of bac-

terial communities in this species (Linnenbrink et al.

2013). Even though gastrointestinal bacteria may have not

necessarily been the pathogenic agents selecting for

immunological divergence in the two subspecies, we may

expect similar geographic or subspecies-specific variation

also among other microbes. Genetic differences between

non-bacterial parasites of the two house mouse subspecies

and the lack of their significant introgression in the

hybrid zone have been described recently (Kv�a�c et al.

2013).

In this study, we have analyzed free-living specimens of

the two European Mus musculus subspecies across a wide

geographic range to answer the question whether the dis-

tinct recent evolutionary histories of the subspecies have

left any footprints in Tlr4 variation. Based on preliminary

data from classical laboratory strains (CLS) and wild-

derived strains (WDS), we expect significant differences

between the two house mouse subspecies. These poten-

tially contrasting patterns could be explained either by

different selection forces mediated by pathogens or simply

by differences in demographic histories of the taxa (e.g.,

population expansions and/or bottlenecks). Given scarcity

of data on pathogen background in the sampled regions,

we tested the two plausible explanations by analyzing also

the mitochondrial cytochrome b (mt-Cytb) gene widely

used as a selectively neutral marker for assessing demo-

graphic histories of species and populations. Whereas

similar patterns observed in both mt-Cytb and Tlr4 would

support the effect of demographic changes, distinct pat-

terns in the two genes would suggest the effect of selec-

tion on Tlr4. By genotyping Tlr4 and mt-Cytb in 44 Mmd

and 30 Mmm sampled across the Western Palaearctic

region, we document (1) a subspecies-specific distribution

of genetic variation, (2) different selection patterns oper-

ating on Tlr4 gene in the two subspecies, and (3) impor-

tant role of recombination increasing the polymorphism

of the Tlr4 gene.

Materials and Methods

Sampling

We sampled 28 and 42 populations (1–2 individuals per

site) of free-living Mmm and Mmd, respectively, scattered

across the Western Palaearctic region (with exception of

two localities from central Asia; Fig. 1, Table S1). In

addition, we included also mice of three CLSs of predom-

inantly Mmd origin (C3Ha, A/J, C57BL/6J; see Yang et al.

(2011) for their genomic composition), 15 WDSs of the

Mmd origin, and nine WDSs of the Mmm origin (Pi�alek

et al. 2008; Vyskocilov�a et al. 2009; for the origin of

WDSs, see Table S1). In comparison with laboratory

strains, WDSs encompass more natural polymorphism

and, at the same time, the homozygote variants are useful

for distinguishing heterozygote sequences of natural pop-

ulations (Gu�enet and Bonhomme 2003; Stephan et al.

2007; Pi�alek et al. 2008).

Assignment of specimens to subspecies

Assigning each analyzed individual to one of the two sub-

species was based on the combination of X-linked and

mtDNA diagnostic markers proven to display low levels

of introgression across the European house mouse hybrid

Figure 1. Distribution of samples analyzed in

this study. Blue circles: Mus musculus

domesticus (Mmd); yellow diamond, red

squares, violet triangles and pink circles:

M. m. musculus (Mmm). Different symbols

represent distinct protein variants of the

ligand-binding region (LBR). Individuals were

assigned to the subspecies using hybrid index

based on five X-linked loci. Dashed line

represents the hybrid zone between two

subspecies. Besides sampling localities of free-

living populations, the localities of wild-derived

strains origin are shown.

ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3

A. Fornuskova et al. Contrasting Patterns of Polymorphism

zone (�Dureje et al. 2012). The first set of markers con-

sisted of five X-linked SINE and/or LINE insertions cho-

sen to be distributed along the whole chromosome: X332,

X65, X347, Btk, and Syap1 (Machol�an et al. 2011). For

each individual, a hybrid index (HI) was calculated as the

mean frequency of Mmm-specific alleles over all five loci

(10 for a female and five for a male). While the majority

of mice displayed HI = 1.00 (Mmm) or HI = 0.00

(Mmd), 13 individuals were not fixed for all Mmm or all

Mmd alleles (Table S1). This may be due to introgression

and/or incomplete lineage sorting of the X-linked mark-

ers. Note that also C57BL/6, that is, one of the most

“classical” laboratory strains of predominately Mmd ori-

gin (Yang et al. 2011), harbors Mmm alleles (Table S1).

However, regardless underlying causes, in all these cases,

admixture was negligible, allowing reliable subspecific

identification.

The mitochondrial marker was a BamHI restriction site

in the Nd1 gene shown to discriminate between the sub-

species (Bo�z�ıkov�a et al. 2005). Mice were assigned to

Mmm when the site was absent and to Mmd when the

site was present. All 62 mice (wild, CLS, and WDS)

assigned on the basis of the HI to Mmd also carried the

BamHI restriction site. Of the remaining 39 mice assigned

to Mmm according to the HI, three individuals (two

wild individuals from Lindhorst and Lauchhammer in

Germany, and one from a still not fully inbredized WDS

established from Lindhorst) carried the Mmd-specific

restriction site, suggesting introgression of Mmd mtDNA

across the hybrid zone into Mmm range (Table S1).

These three specimens (SLINT-WDS, SK843 and SK837)

were analyzed as Mmm in the Tlr4 dataset and as Mmd

in the mtDNA dataset (see below for details).

Genetic variation within subspecies

In total, 101 specimens (free-living mice together with

WDSs and CLSs) were successfully sequenced for both

Tlr4 and mt-Cytb genes. We sequenced exon 3 of Tlr4

(2244 bp), encompassing 90% (748 of 835 amino acid

residues) of the gene coding sequence, following the

protocol described in Fornuskova et al. (2013). Almost

complete mt-Cytb (1123 bp) was sequenced after amplifi-

cation by universal primers L14724 and H15915 (Leco-

mpte et al. 2002). Sequences were manually checked and

aligned using SEQSCAPE v.2.5 (Applied Biosystems, For-

ster city, CA) and BIOEDIT v.7.1.3 (Hall 1999).

Individual Tlr4 alleles (thereafter called haplotypes for

simplicity of comparison with mt-Cytb) were recon-

structed from the complete alignment using the Bayesian

PHASE routine implemented in DnaSP v.5.10 (Stephens

and Donnelly 2003; Librado and Rozas 2009). This analy-

sis was carried out using 1000 iterations, 10 thinning

intervals and 1000 burn-in iterations. Four heterozygous

Tlr4 sequences resolved with low support were checked

by cloning using the protocol of pGEM�-T Easy Vector

System II (Promega Madison, WI). Initially, two clones

from each individual were sequenced and this number

was later increased until we obtained both sequences of

each heterozygote (identification of the four cloned cases

can be found in Table S1). Positions of TLR4 domains

(ECD, TMD, ICD/TIR) were determined using the on-

line program SMART according to Fornuskova et al.

(2013). Amino acids were numbered according to a Gen-

Bank M. musculus TLR4 protein sequence (GenBank

Number: AGA16686.1).

The numbers of nucleotide haplotypes (N) and amino

acid variants (A) for both Tlr4 and mt-Cytb genes were

estimated using Fabox DNA collapser (Villesen 2007).

Nucleotide diversity (p), average number of nucleotide

differences (k), number of polymorphic sites (S) and hap-

lotype diversity (Hd) were computed in DnaSP v.5.10.

Haplotypes were assigned to haplogroups (HG) based on

their phylogenetic interrelationships inferred with MrBa-

yes v. 3.1 (Huelsenbeck and Ronquist 2001) and accord-

ing to a median joining network constructed with

Network v. 4.6.1.1. (Bandelt et al. 1999). The HKY+Γ(Hasegawa et al. 1985) and GTR+Γ (Tavar�e 1986) mod-

els, determined using jModelTest v. 0.1.1. (Posada 2008),

were applied to Tlr4 and mt-Cytb data, respectively. For

both genes, we ran 10,000,000 MCMC generations of

which 2,500,000 generations were discarded as burn-in.

Geographical distribution of the HG was projected onto a

map using the PanMap software (http://www.pangaea.de/

software/PanMap/). All these computations were based on

a subset of wild and WDS mice (i.e., we excluded all

sequences from CLSs).

Analysis of molecular evolution of Tlr4

For detection of recombination breakpoints in the Tlr4

gene, we used two algorithms, the single breakpoint

recombination (SBP) and genetic algorithm recombina-

tion detection (GARD), provided on the DataMonkey

web server (Pond and Frost 2005a,b; Pond et al. 2006a,b;

Delport et al. 2010). The Tlr4 dataset was partitioned

according to the breakpoints detected with the SBP and

GARD methods. Because it is now widely recognized that

the evolutionary process is not homogeneous across sites,

we performed also an analysis partitioned by three codon

positions.

Selection on Tlr4 was analyzed at the intersubspecific

level. We aimed to identify codons subject to positive or

negative selection using test implemented in the Data-

Monkey program (Pond and Frost 2005a; Pond et al.

2006a): random effects likelihood (REL). The REL test



tends to be somewhat susceptible to Type 1 errors, espe-

cially for small datasets, where parameter estimates are

likely to have large associated errors (Pond and Frost

2005b). The Bayes factor was set up to 50. Finally, we

employed the McDonald–Kreitman test (MKT), which

compares variation within species to the amount of diver-

gence between species at putatively neutral (synonymous)

and nonsynonymous sites (McDonald and Kreitman

1991). Four types of comparisons were used in the MKT:

Mmm/Mmd versus rats of the tribe Rattini; Mmm/Mmd

versus R. norvegicus; Mmm/Mmd versus southeastern-

Asian mouse species M. caroli, M. cooki, M. cervicolor;

and Mmm versus Mmd (results available upon request).

All selection tests were applied to a set of wild and WDS

mice (i.e., excluding CLSs). Sequences of Asiatic species

of Mus and Rattini were taken from Fornuskova et al.

(2013).

The crystal structure of mouse TLR4 ECD (PDB 2z64)

was adopted and modified from the RCSB PDB Protein

Data Bank (http://www.rcsb.org/pdb/explore.do?structure-

Id=2z64; Kim et al. 2007). Subsequently, nonsynonymous

substitutions, sites under positive and negative selection

detected by REL, and previously described binding sites

for LPS and MD-2 (lymphocyte antigen 96; Kim et al.

2007; Park et al. 2009; Ohto et al. 2012) were visualized

using PyMOL, v. 1.6 (The PyMOL Molecular Graphics

System, Schr€odinger, LLC; available at http://www.pymol.

org/, accessed January 25, 2013).

Results

Genetic diversity of Tlr4

We successfully amplified Tlr4 sequences of 44 wild Mmd

(27 homozygotes and 17 heterozygotes) and 30 wild

Mmm (17 homozygotes and 13 heterozygotes; see

Table S1 for the number of heterozygous sites for each

individual). We found neither heterozygotes between

Mmd and Mmm subspecific variants nor trans-subspecific

polymorphism. Phylogenetic analysis of amplified

sequences of both genes (Tlr4 and mt-Cytb) showed

divergence of genetic diversity into two clades corre-

sponding to the Mmm and Mmd subspecies (Table S1,

Figs. S1, S2). In total, we found 18 and 15 Tlr4 haplo-

types for Mmm and Mmd, respectively (including WDSs,

CLSs and wild mice). Similarly, we identified 23 and 37

haplotypes of mt-Cytb, for Mmm and Mmd, respectively.

All Mmd with the present BamHI restriction site har-

bored an Mmd-related mt-Cytb haplotype, and the same

holds for Mmm mice (Table S1, Figs. S1B, S2B).

Genetic variation in the Tlr4 locus was considerably

higher in Mmm (NMmm = 18, AMmm = 15, pMmm =0.0025 � 0.00016 SD) than in Mmd (NMmd = 15,

AMmd = 7, pMmd = 0. 0009 � 0.00007 SD). This is even

more noticeable for the ECD with fourfold nucleotide

diversity and twofold number of segregating sites in

Mmm relative to Mmd (Table 1). Contrary to Tlr4,

Table 1. Genetic diversity of Tlr4 and mt-Cytb in two house mouse subspecies.

N/N1 A/A1 p � SD1 k1 S1 Hd � SD1

Tlr4-exon 3 2244 bp

Mmd 15/14 7/6 0.0009 � 0.00007 1.929 10 0.736 � 0.052

Mmm 18/16 15/13 0.0025 � 0.00016 5.595 18 0.882 � 0.028

Tlr4-ECD 1644 bp

Mmd 9/8 5/4 0.0005 � 0.00007 0.845 6 0.554 � 0.066

Mmm 12 7/7 0.0020 � 0.00015 3.267 12 0.800 � 0.043

Tlr4-LBR 666 bp

Mmd 2/1 2/1 0.0000 0.000 0 0.000

Mmm 7/7 4/4 0.0022 � 0.00022 1.473 6 0.627 � 0.063

Tlr4-ICD 531 bp

Mmd 5/5 2/2 0.0020 � 0.00014 1.085 4 0.568 � 0.039

Mmm 8/7 8/7 0.0026 � 0.00016 1.398 4 0.784 � 0.028

Tlr4-TIR 435 bp

Mmd 4/4 2/2 0.0024 � 0.00015 1.052 3 0.551 � 0.038

Mmm 3/2 3/2 0.0001 � 0.00010 0.047 1 0.047 � 0.044

Cyt b 1123 bp

Mmd 37/36 15/15 0.0046 � 0.00022 5.105 49 0.983 � 0.009

Mmm 23/20 9/9 0.0047 � 0.00046 5.254 36 0.974 � 0.016

Mmd, Mus musculus domesticus; Mmm, Mus musculus musculus; 62 and 39 specimens were analyzed for Mmd and Mmm, respectively; N, num-

ber of nucleotide haplotypes; A, number of amino acid variants; p, nucleotide diversity; k, average number of nucleotide differences; S, number

of polymorphic sites; Hd, haplotype diversity; SD, standard deviation.1Indicate analysis without wild-derived strains and classical laboratory strains.



genetic variation in mt-Cytb was comparable for both

subspecies (NMmm = 23, NMmd = 37; pMmm = 0.0047 �0.00046 SD, pMmd = 0.0046 � 0.00022 SD; Table 1).

Moreover, in all but one Mmd samples, we identified a

single protein variant of the LBR. The only exception was

the A/J laboratory strain which possessed the conservative

substitution V254I. This lack of polymorphism is in con-

trast to variation in Mmm where four different variants

of LBR were found, with two of them being equally fre-

quent in the Mmm distribution area (Fig. 1). These vari-

ants differed at three codons (F350C, D462N, and I464V;

Table 2). Nevertheless, all substitutions in the LBR

brought about exchanges between biochemically similar

amino acids. An overview of all amino acid substitutions,

their physicochemical properties and distribution are pre-

sented in Fig. 2 and Table 3.

Haplotype network analysis and distributionof genetic groups

The haplotype networks based on nucleotide sequences of

exon 3 of Tlr4 were strikingly different in the two mouse

subspecies. In Mmd, there was a single most frequent

haplotype (H_18; Fig. 3A). It was present in 71% of all

individuals (including CLSs and WDSs) and in 66% of

wild mice only (in wild mice it was present in 18 speci-

mens in the homozygote state and in 11 specimens as

heterozygotes). Conversely, in Mmm, individual haplo-

types were more evenly represented, none of them occur-

ring in more than 39% of all specimens. The most

common Mmm haplotype (H_5) was found in 33% of

wild mice only. Based on the phylogenetic analysis and

topology of the haplotype network (Figs. S1, S2), we

defined one and two HG for each subspecies, respectively

(HG-Idfor Mmd and HG-Im and HG-IIm for Mmm).

Notwithstanding the absence of distinct genetic structur-

ing of HG-Id, a subgroup of three haplotypes (for clarity

hereafter denoted as S-I) appears rather basal to other

two subgroups (S-II and S-III, respectively; Fig. 3A) and

Table 2. Description of ligand-binding region (LBR) variants. Colored

symbols correspond to Fig. 1.

LBR variants I254V F350C D462N I464V

LBR-V-1d V F D I

LBR-V-2d A/J I F D I

LBR-V-1 m V F D I

LBR-V-2 m V F N I

LBR-V-3 m V C D I

LBR-V-4 m V F D V

The distribution of particular variants among sampled specimens is

shown in Table S1.

Table 3. Physicochemical properties of the amino acids involved in

nonsynonymous substitutions of Tlr4.

Position aa1 Properties aa2 Properties

122 S SM, P, NEU C SM, NP, NEU

160 F NP, NEU L NP, NEU

209 I NP, NEU M NP, NEU

2541 V NP, NEU I NP, NEU

3501 F NP, NEU C SM, NP, NEU

4621 D SM, P, NEG N SM, P, NEU

4641 I NP, NEU V NP, NEU

593 D SM, P, NEG E P, NEG

637 I NP, NEU V NP, NEU

668 G SM, NP, NEU E P, NEG

670 S SM, P, NEU C SM, NP, NEU

7612 R P, POS H P, POS

7992 P SM, NP, NEU A SM, NP, NEU

8112 K P, POS N SM, P, NEU

831 M NP, NEU T SM, P, NEU

SM, small; NP, nonpolar; P, polar; NEU, neutral; POS, positively

charged; NEG, negatively charged.1Sites placed in ligand-binding region.2Sites placed in Toll/interleukin-1 domain.

Figure 2. Overview of Tlr4 nonsynonymous substitutions in Mmd and

Mmm. Numbers above alignment indicate amino acid position. ECD,

extracellular domain; TMD, transmembrane domain; ICD, intracellular

domain; LBR, ligand-binding region; TIR, Toll/interleukin-1 domain.

Distribution of individual haplotypes (=alleles) among sampled

specimens is presented in Table S1.



restricted to the eastern Mediterranean region and north-

ern Tunisia, while haplotypes of the latter two subgroups

either have a wide distribution (e.g., H_18, H_24) or have

arisen in situ after westward spread of ancestral haplo-

types (see the inset in Fig. 3A). This geographic distribu-

tion suggests a recent expansion accompanied by a loss of

variation. This is especially exemplified by the star-like

pattern of S-III haplotypes centered on haplotype H_18

(Fig. 3A).

In Mmm, there were two distinct haplotype clouds sep-

arated at least by eight substitutions (HG-Im and HG-

IIm). Both groups were interconnected by H_2 (CZ,

Bu�skovice) and H_19 (WDS, DE, Lindhorst) which were

not included in any HG (see below). The geographical

distribution of HG-Im and HG-IIm is very wide, from

central Asia to central Europe and they are largely over-

lapping in most of the Mmm distribution area. Interest-

ingly, the distance between HG-Im and HG-IIm is higher

(minimum eight substitutions) than the distance between

HG-Im and HG-Id (minimum four substitutions). In

contrast to Tlr4, the pattern of the mt-Cytb haplotype

network was very similar for both subspecies with sev-

eral star-like branching patterns suggesting local spatial/

demographic expansions (Fig. 3B). The geographic distri-

bution of both Mmm and Mmd HG seems to be more

intermingled than that of Tlr4 HGs (see the inset in

Fig. 3B). Identification of haplotypes in particular speci-

mens is detailed in Table S1.

(A)

(B)

Figure 3. (A) Haplotype network and

haplogroup distribution of Tlr4, H_haplotypes,

HG-, haplogroups, S- subgroups identified in

Figs. S1a and S2a. The size of circles

corresponds to the frequency of haplotypes;

length of lines is related to the number of

substitutions. More detailed information can

be found in Table S1. The inset figure

represents the geographical distribution of

HGs. Color circles on the map represent the

proportion of particular HG or S (colors

correspond to the haplotype network), labels

indicate geographic assignment to population

groups detailed in Table S1; dashed line shows

the position of the house mouse hybrid zone.

H_2 and H_19 were excluded from HG due to

recombination (see the text for more details).

(B) Haplotype network and haplogroup

distribution of mt-Cytb, H_ identified

haplotypes, HG-, identified haplogroup. The

size of circles corresponds to the frequency of

haplotypes; length of lines is related to the

number of substitutions. More detailed

information can be found in Table S1. The

inset figure represents the geographical

distribution of HGs. Color circles on the map

represent the proportion of particular HG

(colors correspond to the haplotype network);

labels indicate geographic assignment to

population groups detailed in Table S1; dashed

line shows the position of the house mouse

hybrid zone.



Recombination and selection in the Tlr4gene

A recombination breakpoint between Mmd and Mmm at

position 1779 bp was detected by both tests implemented

in DataMonkey. This breakpoint was recognized in

one Mmm individual (ST8335, H_13) sampled in

Poland. However, it is based only on a single synonymous

substitution at position 849 and homoplasy seems equally

plausible explanation. At the intrasubspecific level, we

detected recombination in two individuals of Mmm. This

breakpoint was identified in a conserved region between

the LBR and ICD (the SBP algorithm located the recom-

bination breakpoint to nucleotide position 1587 = AA

529, while GARD placed it to position 1611 = AA 537).

Haplotypes H_2 and H_19 likely represent recombinant

haplotypes between two main Mmm HG (Fig. S3).

The REL test detected eight positively and 14 negatively

selected sites (Table 4). Four of the positively selected

sites were placed in the ECD; however, none of them was

in the LBR (Fig. 4, Table 4). Ten of the 14 negatively

selected sites were located in the ECD, three of these co-

dons being in LBR (Fig. 4, Table 4). The MK test

revealed mostly signs of negative selection (not shown).

Discussion

Tlrs are generally believed to evolve mainly under purify-

ing selection and, thus, it has been predicted that these

genes are relatively uniform within species (e.g., Mukher-

jee et al. 2009). Contrary to this expectation, we found a

moderate intrasubspecific level of Tlr4 polymorphism.

With 15 protein variants in Mmm and 7 protein variants

in Mmd, this finding holds true more for Mmm than for

Mmd. Indeed, we revealed decreased variation in Mmd

Tlr4 both at the nucleotide and amino acid levels

(Table 1), especially in the LBR where we found only a

single variant in Mmd, whereas higher polymorphism

level (four variants of LBR) is maintained in Mmm popu-

lations. Given the crucial function of TLR4 in mammalian

innate immune defense, we may assume that the single

LBR variant of TLR4 was advantageous in the past, before

or during expansion of Mmd into Western Mediterranean

and western/north Europe. On the other hand, we

observed similar levels and geographic patterns of genetic

variation of mt-Cytb in both subspecies. This indicates

that the observed pattern does not result from a generally

Figure 4. Ribbon diagram of the TLR4 extracellular domain (ECD) 3D

structure (PDB 2z64 from RCSB PDB Protein Data Bank, http://www.

rcsb.org/pdb/explore.do?structureId=2z64, functional sites were

described according to (Kim et al. 2007; Park et al. 2009; Ohto et al.

2012) and important substitutions were visualized as amino acid

space-fill models: cyan corresponds to binding positions for MD-2,

yellow represents binding sites for lipopolysaccharides (LPS), red

represents nonsynonymous amino acid changes, orange represents

sites under negative selection detected between subspecies by

random effects likelihood (REL); black stars represent detected sites

under positive selection differing between the subspecies as revealed

by REL; the TLR4 ECD is represented by green color, MD-2 is

represented by cyan color, LBR, Ligand-Binding Region is marked by

dashed lines. Description of sites responsible for LPS binding and MD-

2 binding is in Table S2, modeling of different sites and design

correction made in PyMOL Version 1.5.

Table 4. Selection tested by random effects likelihood (REL) in both subspecies together, including wild-derived strains (WDSs); classic laboratory

strains (CLSs) were excluded for this analysis.

REL (Mmm+Mmd+WDSs) ECD (88–635) TMD (636–658) ICD (659–835)

Positively selected sites 122, 160, 209, 593 637 670, 811, 831

Negatively selected sites 104, 132, 139, 192, 370, 416, 463, 529, 537, 575 647 690, 719, 833

ECD, extracellular domain, TMD, transmembrane domain, ICD, intracellular domain. Underlined sites in ECD are placed in ligand-binding region

(248–469). Underlined sites in ICD are placed in Toll/interleukin-1 domain (671–816). Numbers in brackets indicate position of domains in protein

(ECD start with codon 88, first 87 codons are in exon 1 and 2). All sites detected by REL had pp = 0.99.



decreased level of genetic polymorphism in Mmd. Alto-

gether, our results may imply the action of contrasting

types of selection acting specifically on Tlr4 in the two

house mouse subspecies. A similar contrast in selection

on TLR4 across geographically distinct populations is

known also in other species. For instance, in humans, it

has been shown that different haplotypes are positively

selected in Sub-Saharan Africa and Eurasia (Ferwerda

et al. 2007). Identifying selective forces differentiating

subspecies and populations thus appears an intriguing

question of current evolutionary biology.

The role of TLR4 in LPS signaling is indisputable and

molecular mechanisms of LPS binding were very well

described in human and/or mouse (Kim et al. 2007; Park

et al. 2009; Resman et al. 2009; Ohto et al. 2012). LPSs

are present in the outer membrane of Gram-negative bac-

teria and immunologically act as endotoxins, that is, sub-

stances eliciting a strong immune response in animals.

Variability of LPS may affect adhesive properties of a

microorganism to the cells of its host but also the

induced release of inflammatory mediators. Modifications

of LPSs (mainly acylation in the lipid A region) play an

important role in the infection process, evasion of the

host immune response, and serotypification of Gram-neg-

ative bacteria (Robinson et al. 2008). Polymorphism of

LPSs has been already shown to be associated with differ-

ences in virulence of bacterial strains, for example, Franci-

sella tularensis, Pseudomonas aeruginosa or Yersinia pestis

(Day and Marrceau-Day 1982; Ray et al. 1991; Hajjar

et al. 2006; Knirel et al. 2006; Montminy et al. 2006), and

as such may be responsible also for evolution and mainte-

nance of recognition mechanisms. This applies especially

to Tlr4 variation. As the genetic variation of human and

livestock TLR4 is associated with susceptibility to various

infectious and inflammatory diseases (e.g., Leveque et al.

2003; Hawn et al. 2005; Achyut et al. 2007; Sentitula

Kumar and Yadav 2012; Zaki et al. 2012) and several

nonsynonymous single nucleotide substitutions (nsSNP)

has been identified as immunologically relevant (Ferwerda

et al. 2007), we focused on physical properties of the

nsSNPs we detected in the house mouse Tlr4. In total, we

detected 15 nsSNP positions, which were distributed

evenly across the whole sequenced region including the

ECD, TMD, and ICD. Of these 15 nsSNPs we found four

(V254I, F350C, D462N and I464V) that were located in

the LBR close to the ligand-binding site of LPSs (Fig. 4).

Out of these, the substitution V254I has been identified

only in the LBR of the A/J laboratory strain and not in

any WDS and/or free-living mice (see also Smirnova et al.

2000). We, therefore, suggest that this substitution does

not represent a naturally occurring polymorphism and

may have originated in laboratory breeds. On the other

hand, particularly functionally important might be the

residues 462 and 464 that lie in immediate topological

proximity to site F461, which has been previously identi-

fied as a residuum essential for LPS binding through

hydrophobic interactions in mammals (Park et al. 2009;

Resman et al. 2009). We, therefore, hypothesize that these

nsSNPs can influence the protein function. Our tests of

selection, however, did not support this view as no posi-

tively selected sites were identified in the LBR. This sug-

gests that D462N and I464V substitutions either have no

functional impact or, at least, that there is no selection dif-

ferentiating these sites in Mmm and Mmd. Nonetheless,

the selection analysis showed that three of eight sites posi-

tively selected on the intersubspecific level were present in

the MD-2-binding region, indicating selection differenti-

ating Mmm and Mmd in the TLR4-MD-2 co-evolution.

Recent data have shown that mouse subspecies harbor

genetically different parasites (e.g., Cryptosporidium tyzze-

ri; Kv�a�c et al. 2013). Both subspecies may therefore differ

in immune response to specific genetic lineages of patho-

gens. Preliminary laboratory experiments have already

shown differences in immunological response between

two WDSs derived from both subspecies (Mmm BULS

and Mmd STRA) by stimulating in vitro by Concanava-

line A and a B-cell mitogen bacterial LPS (Pi�alek et al.

2008).

Although most substitutions identified in the present

study involve physically very similar amino acids, it has

been shown that even subtle changes in the topological

proximity of the binding interface may have substantial

impact on the protein function and binding affinity

(Zhang et al. 2012). Further studies are, however, needed

to test the functional significance of the nsSNPs for rec-

ognition of LPS variants.

Previous studies showed that genes encoding TLRs exhi-

bit moderate levels of polymorphism even at intraspecific

level (Smirnova et al. 2000; Tschirren et al. 2011; Bergman

et al. 2012; Grueber et al. 2012) and that this can have

important fitness consequences. In free-living populations,

it was documented that selection linked with presence of

pathogens can vary across different geographic regions and

over time (Tschirren et al. 2012). Polymorphism in

immune receptors is thought to be primarily maintained

by pathogen-evoked balancing selection. This may be

viewed as an evolutionary key-and-lock process described

by the Matching alleles model (Frank 1993). Applied to

receptor-ligand co-evolution, this model proposes that

polymorphism in ligands protecting parasites from recog-

nition is mirrored by adaptive host polymorphism allow-

ing detection of ligand-variants by specifically matching

receptor alleles (Agrawal and Lively 2002, 2003).

In addition to nucleotide substitutions, also intragenic

recombination can very quickly create new allele variants.

In house mouse, the effect of recombination in the



evolution of immune genes is well documented, for

example, in the MHC genes (Cizkova et al. 2011). How-

ever, in most recent studies on intraspecific TLR poly-

morphism the relevant tests of recombination have not

been performed. Using two alternative approaches, our

study detected recombination events in the ECD located

close to the boundary with the TMD in Mmm. This find-

ing adds another piece of information to the puzzle of

PRR polymorphism in free-living rodents showing that

recombination might be an important factor increasing

TLR variability. Our results are consistent with studies of

several other mammals reporting signals of recombination

in the ECD in human TLR4 (Zaki et al. 2012) or bovine

TLR3, TLR4 and TLR10 (Seabury et al. 2010). Detailed

analysis of our sequences suggests that haplotypes H_2

and H_19 are recombinants composed of the ECD from

haplogroup HG-IIm and ICD of HG-Im. These two

Mmm haplotypes are genetically dissimilar and were

found in two specimens separated by 500 km (see

Table S1). Assuming that they represent two independent

recombination events, we suggest that recombination in

this genic region may be relatively frequent in nature. On

the other hand, the recombinant haplotypes were found

only in two individuals and the estimation of real selec-

tive advantage of recombination remains unknown.

Because the recombination breakpoints combine different

ECDs and ICDs, the WDS SLINT bearing H_19 (in com-

bination with other WDSs from Tlr4 haplogroups HG-Im

and HG-IIm) provides a unique opportunity to discrimi-

nate the role of LBR-mediated LPS recognition from the

transduction of the signal by the TIR domain.

Although pathogens likely play an important role in

evolution of Tlr4 variability, it may be admitted that the

observed difference between the subspecies in TLR4 poly-

morphism might have arisen as a result of nonadaptive

evolutionary processes during mouse colonization of the

Western Palearctic. For example, in some avian popula-

tions affected by bottlenecks the dominant force influenc-

ing evolution of TLRs seems to be genetic drift,

outweighing the effect of selection (Grueber et al. 2012,

2013). Similarly, genetic drift also shaped the genetic his-

tory of human TLR4 during population expansion out of

Africa (Netea et al. 2012). Thus, the pattern observed in

mice might result, for example, from differences between

subspecies in historical demographic processes (quick

expansion of Mmd and two founder populations or ref-

uges for Mmm). In such a case we would, however,

expect similar contrasting patterns in mt-Cytb. As this

was not the case, we may consider the explanation of the

observed pattern of Tlr4 by genetic drift as unlikely.

Finally, we must also bear in mind that Mmd Tlr4 may

not be the positively selected gene itself but only a gene

involved in gene hitchhiking. Nevertheless, this hypothesis

is in contradiction with results of selection analysis, which

have detected eight positively selected sites in ECD of

free-living Mus musculus.

Acknowledgment

We thank the following colleagues for donating mouse

samples: S. J. E. Baird, J. Go€uy de Bellocq, N. Bulatova, J.

Burgstaller, D. �C�ı�zkov�a, B. Dod, L’. �Dureje, J. Forejt, S.

Gryseels, H. C. Hauffe, S. Ihle, A. Kone�cn�y, S. Mart�ınek,

N. Mart�ınkov�a, F. Matur, P. Mikul�ı�cek, G. Mitsainas, A.

Mishta, M. Mrkvicov�a, P. Munclinger, J. Pi�alkov�a, A. Ri-

bas, J. Svobodov�a, V. Volobouev, M. Vujo�sevi�c, J. M.

W�ojcik, and J. Zima. L. Vl�ckov�a genotyped X-linked loci

and the Nd1 gene. This research was supported by the

Czech Science Foundation (project 206/08/0640) to JP

and MM and by Ministry of Education, Youth and Sports

of the Czech Republic, NextGenProject: Next-generation

technologies in evolutionary genetics (CZ.1.07/2.3./

20.0303). We thank S. J. E. Baird for language corrections

and significant improvement of previous versions of the

manuscript and J.-F. Cosson for general support and

commentaries. We also thank two anonym reviewers for

their constructive suggestions. Phylogenetic reconstruc-

tions were computed at the CBGP HPC computational

platform located at Centre de Biologie et Gestion des

Populations, Montferrier-sur-Lez.

Conflict of Interest

None declared.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Summary of sampled specimens, identification

of haplotypes, and NCBI GenBank accession numbers.

Table S2. Binding sites between TLR4/LPS/MD-2.

Figure S1. (A) Tlr4, Phylogeny based on Bayesian infer-

ence. (B) mt-Cytb, Phylogeny based on Bayesian infer-

ence.

Figure S2. (A) Tlr4, Haplogroup definition. (B) mt-Cytb,

Haplogroup definition.

Figure S3. Evidence of recombination between HG-Im

and HG-IIm of Mmm.



contrasting patterns of polymorphism and selection in

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